Relationship between elongation factor I- and elongation factor II- dependent guanosine triphosphatase activities of ribosomes. Inhibition of both activities by ricin.
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ABSTRACT:
SUBMITTER: Sperti S
PROVIDER: S-EPMC1165562 | biostudies-other | 1975 Jun
REPOSITORIES: biostudies-other
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Project description:The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.
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Project description:Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.
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Project description:Ricin and modeccin do not affect the total number of ribosomes to which phenylalanyl-tRNA becomes bound in the EF 1-dependent reaction. Previous inconsistencies resulted from the use of the nitrocellulose-filter technique, which overestimates the number of control ribosomes engaged in the binding reaction if trace amounts of EF 2 contaminate the ribosomal preparations.
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Project description:Protein release factor 3 (RF3), a guanosine triphosphatase, binds to ribosome after release of the nascent peptide and promotes dissociation of the class I release factors during the termination of protein synthesis. Here we present the crystal structure of the 70S ribosome with RF3 in the presence of a nonhydrolyzable GTP analogue, guanosine 5'-β,γ-methylenetriphosphate (GDPCP), refined to 3.8 Å resolution. The structure shows that the subunits of the ribosome are rotated relative to each other compared to the canonical state, resulting in a P/E hybrid state for the transfer RNA. The substantial conformational rearrangements in the complex are described and suggest how RF3, by stabilizing the hybrid state of the ribosome, facilitates the dissociation of class I release factors.
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Project description:Even though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of an Escherichia coli strain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Altogether, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.
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Project description:Translation elongation in eukaryotes is mediated by the concerted actions of elongation factor 1A (eEF1A), which delivers aminoacylated tRNA to the ribosome; elongation factor 1B (eEF1B) complex, which catalyzes the exchange of GDP to GTP on eEF1A; and eEF2, which facilitates ribosomal translocation. Here we present evidence in support of a novel mode of translation regulation by hindered tRNA delivery during mitosis. A conserved consensus phosphorylation site for the mitotic cyclin-dependent kinase 1 on the catalytic delta subunit of eEF1B (termed eEF1D) is required for its posttranslational modification during mitosis, resulting in lower affinity to its substrate eEF1A. This modification is correlated with reduced availability of eEF1A·tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at the elongation level.
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Project description:Chloroplasts possess bacterial-type systems for transcription and translation. On the basis of the identification of a Chlamydomonas reinhardtii gene encoding a RelA-SpoT homolog (RSH) that catalyzes the synthesis of guanosine tetra- or pentaphosphate [(p)ppGpp], we have previously suggested the operation of stringent control in the chloroplast genetic system. Although RSH genes have also been identified in several higher plants, the activities of the encoded enzymes and their mode of action in chloroplasts have remained uncharacterized. We have now characterized the intrinsic (p)ppGpp synthase activity of chloroplast extracts prepared from pea (Pisum sativum). Fractionation by ultracentrifugation suggested that the (p)ppGpp synthase activity of a translationally active chloroplast stromal extract was associated with 70S ribosomes. Furthermore, this enzymatic activity was inhibited by tetracycline, as was the peptide elongation activity of the extract. Structural comparisons between rRNA molecules of Escherichia coli and pea chloroplasts revealed the conservation of putative tetracycline-binding sites. These observations demonstrate the presence of a ribosome-associated (p)ppGpp synthase activity in the chloroplasts of a higher plant, further implicating (p)ppGpp in a genetic system of chloroplasts similar to that operative in bacteria.
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Project description:Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system.
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Project description:Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.
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Project description:1. Ricin (a toxic protein from the seeds of Ricinus communis) is a powerful inhibitor of the poly(U)-directed incorporation of phenylalanine into polypeptides catalysed by isolated rat liver ribosomes and elongation factors 1 and 2 (EF 1 and EF 2). The inhibition can be largely overcome by increasing the concentration of ribosomes. 2. The toxin does not affect the binding of phenylalanyl-tRNA to ribosomes catalysed by EF 1, nor does it inhibit the puromycin reaction used as a test for peptide-bond formation catalysed by ribosomes. 3. Ricin inhibits the ribosome-linked GTP hydrolysis catalysed by EF 2. 4. Ribosomes treated with ricin and washed through sucrose gradients containing 0.6m-NH(4)Cl are functionally inactive in those assay systems that are sensitive to the presence of added toxin. 5. It is suggested that ricin brings about an irreversible modification of ribosomes which impairs their ability to interact with EF 2. Since ricin inhibits at a molar concentration much lower than that of ribosomes it probably acts catalytically. No added cofactor is necessary for the inhibitory action of the toxin.
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